| Description | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | Product IntroductionAlamar Blue detection reagent provides a simple, rapid, reliable and safe method for cell proliferation and cytotoxicity detection, which is suitable for high-throughput detection experiments. The main component of the detection reagent is a redox indicator. In the oxidized stateProduct IntroductionAlamar Blue detection reagent provides a simple, rapid, reliable and safe method for cell proliferation and cytotoxicity detection, which is suitable for high-throughput detection experiments. The main component of the detection reagent is a redox indicator. In the oxidized state, it appears purple-blue and non-fluorescent, while in the reduced state, it turns into a reduction product with pink or red fluorescence, with an absorption peak of 530-560nm and an emission peak of 590nm.In the process of cell proliferation, the ratios of NADPH/NADP, FADH/FAD, FMNH/FMN and NADH/NAD in the cell increase and are in a reducing environment. The dye taken into the cell is reduced by these metabolic intermediates and cytochromes and then released outside the cell and dissolved in the culture medium, changing the culture medium from non-fluorescent indigo blue to fluorescent pink. Finally, use an ordinary spectrophotometer or fluorophotometer for detection, and the absorbance and fluorescence intensity are proportional to the number of active cells.Instructions1. Add 10µl of detection reagent to 100µl of cell suspension, and incubate in a cell incubator for 2-6 hours. The color of the medium changes from indigo blue to pink and you can proceed to the next step.2. It is recommended to use a fluorescence microplate reader for detection, the excitation light wavelength is between 530-560 nm, the emission light wavelength is 590 nm, and the relative fluorescence unit (RFU) is recorded.3. Draw a standard curve or cell growth curve: the ordinate (Y axis) is the relative fluorescence unit (RFU); the abscissa (X axis) is the cell number or time point or drug concentration.Precautions1. The appropriate density of cells can increase the detection sensitivity. For 96-well plates, we recommend seeding 100 microliters of cells per well. The cell concentration range is: 100-10,000/well for adherent cells, 2,000-50,000/well for suspension cells, and medium as a blank control. For 384-well plates, the cell concentration and seeding volume are both halved.2. The whole process should be aseptic operation, because microbial contaminants can also reduce the detection reagents and affect the experimental results.3. Pay attention to the concentration of inoculated cells and the incubation time after adding detection reagents. If the cell concentration is too high or the incubation time is too long, it will cause a secondary reduction reaction, resulting in colorlessness and disappearance of fluorescence.4. When incubating, avoid light.5. This product can use fluorescence or spectrophotometric detection, but the sensitivity of fluorescence is high, and the experimental error is small. Fluorescence detection is recommended... Read More | Inquire | Glycogen and starch generate glucose-1-phosphate (1PG/G1P) during the process of phosphohydrolysis. This reagent kit provides a simple, sensitive, and rapid determination method: Glucose-1-phosphate (1PG/G1P) is reduced from NADP+to NADPH by the sequential action of phosphoglucose mutase and Glycogen and starch generate glucose-1-phosphate (1PG/G1P) during the process of phosphohydrolysis. This reagent kit provides a simple, sensitive, and rapid determination method: Glucose-1-phosphate (1PG/G1P) is reduced from NADP+to NADPH by the sequential action of phosphoglucose mutase and phosphoglucose dehydrogenase. The content of glucose-1-phosphate (1PG/G1P) in the sample can be calculated by detecting the increase in NADPH at 340nm.Composition and preparation of reagent kit: Reagent name Specifications Save requirements Remarks Extraction solution Liquid 100mL x 1 bottle 4 ℃ storage / Reagent 1 Powder mg x 1 tube 4 ℃ storage Shake or centrifuge the reagent a few times before use to make it fall to the bottom, then dissolve it in 1.2mL of distilled water for later use. Reagent 2 Powder mg x 1 tube Store at -20 ℃ Shake or centrifuge the reagent a few times before use to make it fall to the bottom, then dissolve it in 1.2mL of distilled water for later use. Reagent 3 Liquid 16mL x 1 bottle 4 ℃ storage / Reagent 4 Powder mg x 1 tube Store at -20 ℃ Shake or centrifuge the reagent a few times before use to make it fall to the bottom, then add 1 Dissolve 1mL of distilled water for later use. TRC 1 powder 4 ℃ storage Only used to identify whether the reagents in the kit are normal (not involved in result calculation). Usage: Use a pre standard tube (GIP) to shake the powder a few times until it falls to the bottom, then add 0.5mL of distilled water and mix well to dissolveDilute GIP with a concentration of 4mg/mL and then dilute it four times to 1mg/mL for later use: follow the instructions in the sample addition table for the measuring tube operationRequired instruments and supplies:ELISA reader, 96 well plate, desktop centrifuge, adjustable pipette, mortar, ice and distilled water.Determination of glucose-1-phosphate (1PG/G1P) content:1. Sample preparation① Organizational sample:Suggest weighing around 0 1g of tissue, add 1mL of extraction solution, and homogenize in an ice bath. Centrifuge at 12000rpm, 4 ℃ for 10 minutes, take the supernatant, and place it on ice for testing.[Note]: If the sample size is increased, it can be extracted in a ratio of tissue mass (g) to extraction solution volume (mL) of 1:5-10.② Bacterial/cellular samples:Collect bacteria or cells into a centrifuge tube first, centrifuge and discard the supernatant; Take about 5 million bacteria or cells and add them to 1mLExtract solution, sonicate bacteria or cells (ice bath, power 200W, sonication for 3s, interval 10s, repeated 30 times); Centrifuge at 12000rpm at 4 ℃ for 10 minutes, take the supernatant, and place it on ice for testing.[Note]: If the sample size is increased, extraction can be carried out in a ratio of 500-1000:1 of bacteria/cell quantity (104) to extraction solution (mL).③ Liquid sample: direct detection.2. Machine testing:① Preheat the enzyme-linked immunosorbent assay (ELISA) reader for at least 30 minutes and adjust the wavelength to 340nm.② Thaw the reagent to room temperature (25 ℃);③ Add reagents to the 96 well plate in the following order according to the table:② Thaw the reagent to room temperature (25 ℃);③ Add reagents to the 96 well plate in the following order according to the table: Reagent name (µL） Measurement tube Blank tube (only done once) Reagent 1 10 10 Reagent 2 10 10 Reagent 3 150 170 Sample 20 / Mix well, incubate at room temperature (25 ℃) for 20 minutes, and then read A1 at 340nm (if the A value continues to increase, the incubation time needs to be extended until the absorbance value remains unchanged within 2 minutes). Reagent 4 10 10 Mix well, incubate at room temperature (25 ℃) for 20 minutes, and then read A2 at 340nm (if the A value continues to increase, the incubation time needs to be extended until the absorbance value remains unchanged within 2 minutes). Δ A=(A2-A1) measurement - (A2-A1) blank.[Note] 1 If the difference in Δ A is hovering around zero, the sample size V1 can be increased (such as increasing to 50 µ L, the three phases of the reagent should be reduced while keeping the total volume unchanged), or the sample sampling mass W can be increased. The changed V1 and W need to be substituted into the formula for recalculation.If the A2 value exceeds 1.2, the amount of sample added V1 can be reduced (such as to 10 µ L, the three-phase reagent should be increased while keeping the total volume unchanged), or the sample can be diluted with distilled water (keeping the sample addition system unchanged), and the changed V1 and D need to be substituted into the formula for recalculation.Result calculation:1. Calculated by sample weight:1PG/G1P content (µ g/g fresh weight)=[(Δ A ÷ (ε× d) × V2 × 106 × MR] ÷ (W × V1 ÷ V) × D=836 × Δ A ÷ W × D2. Calculated by the number of cells:1PG/G1P content (µ g/104 cell)=[(Δ A ÷ (ε× d) × V2 × 106 × MR] ÷ (500 × V1 ÷ V) × D=1.7 × Δ A × D. 3. Calculated by liquid volume:1PG/G1P content (µ g/mL)=[(Δ A ÷ (ε× d) × V2 × 106 × Mr] ÷ V1=836 × Δ A ε---NADPH Molar extinction coefficient，6.22×103 L/mol/cm； d---96 Orifice plate optical diameter，0.5cm； V---Add volume of extraction solution，1 mL； V1---Add sample volume，0.02mL V2---Total reaction volume；0.2mL=2×10-4L； W---Sample quality，g； Mr---Glucose-1-phosphate（1PG/G1P）Molecular weight；260； 500---Number of cells, in millions； D---Dilution ratio，Undiluted is 1。 /... Read More | Inquire |